Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Competence-Inducing Factor of Bacillus stearothermophilus

An incompetent mutant (4S Com(-)) does not release competence-inducing factor (CF) into the culture medium and is not infected with TP-1C phage deoxyribonucleic acid (DNA) unless CF is added to the transfection assay. A TP-1C phage-resistant mutant (4S Ton-r) releases relatively large amounts of CF into the culture medium but is not infected with TP-1C phage DNA, even in the presence of CF. The production of CF by the wild type or Ton-r mutant and the ability of the wild type or Com(-) mutant to react with CF does not occur after these cultures have grown at 67 C for 1 hr or longer. A preliminary characterization of the CF is described. The autolytic enzyme or the temperate phage of the wild type and the Ton-r and Com(-) mutants do not have competence-inducing activity.     

Temporary,reversible binding of the competence factor to cellular receptors ofPneumococcus during induction of competence

The binding of the competence factor to cellular receptors of physiologically non-competent cells of Pneumococcus was followed as a function of time. A transformation medium without bovine serum albumin was used to study the binding of the competence factor. Control cells without the added factor remained completely non-competent under these conditions. The maximal binding of the factor to the cellular receptors took place already after 3 min of contact of the cells with the factor at 37 degrees C. After 10 min, when the maximum induction of competence occurs in the system used, the competence factor is fully released from the receptors to the medium. It follows that within the period between the 3rd and 10th min, when the cells are being modified for the irreversible binding of DNA, the presence of the competence factor on the cells is no longer necessary.     

DNA fixation and development of transformability and transfectability in Bacillus subtilis

Summary After pre-competent cells of Bacillus subtilis are placed in the medium in which competence develops, peak competence for transformation is found to occur earlier than competence for transfection by DNA from phages whose DNA is dissimilar to that of the cells. There is a nonlinear dependence of transfection on the concentration of DNA from phage SP 82 despite a linear dependence of DNA fixation on DNA concentration. Both results support the idea that fixation of DNA is a poor indicator of the actual competence of the cells for both transformation and transfection.     

Development of Competence in the Bacillus subtilis Transformation System

Competence in Bacillus subtilis, assayed by the ability of cells to be transformed with bacterial deoxyribonucleic acid (DNA) or transfected by phage DNA, has been shown to occur in a single semisynthetic medium with peak activity occurring 3 hr after the cessation of logarithmic growth. No step-down conditions or culture manipulations were necessary for routine transfection of 1% of the population. The results demonstrate that bacteriophage DNA is a valid assay for studying the development of competence in B. subtilis. Predictions of workers using transforming bacterial DNA, who have suggested that competence in B. subtilis is associated with a specific phase of growth, are substantiated. The peak of competence is not affected by marked differences in the rate of growth during the logarithmic phase. The effect on development of competence by this procedure of some components (including casein hydrolysate, tryptophan, and histidine) which were routinely included in the transformation medium by other investigators has been determined by use of infectious phage DNA as an assay. We have demonstrated that tryptophan, as well as histidine, increases the transformation frequency—even in strains which do not have auxotrophic demands for these components. Glutamic acid and alanine depress optimal levels of transfection.     

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.     

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.     

Transfecting activity of Pseudomonas aeruginosa bacteriophage SM

Factors affecting the efficiency of transfection of Ps. aeruginosa PAO1 cells by the temperate SM bacteriophage DNA have been determined. The efficiency of transfection by DNA preparations isolated from the wild type bacteriophage SMc+ or its thermoinducible mutant SM cts6 is practically the same. The frequency of transfection is (7-9) X 10(4) of infectious centers per mkg of transfecting DNA. Variability in the frequencies of transfection has been registered depending of the infection conditions or on the transfer of the Ps. aeruginosa PAO1 recipient strain population into the competence phase. The efficiency of transfection is increased by the addition of Ca2+ or Mg2+ ions affecting the adsorption and absorbtion of phage DNA by the recipient cells. Optimal concentrations of the bivalent metal ions are 0.15M CaCl2 and 0.2M MgCl2. The results obtained have been used for optimizing the conditions of Ps. aeruginosa PAO1 transfection by SM bacteriophage DNA.     

Bacteriophage N3 of Haemophilus influenzae. IV. Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae cells by N3 phage or transfection by DNA from phage were examined. After infection by whole phage three forms of intracellular phage DNA were observed by sedimentation velocity analysis. These forms are probably twisted circles, open circles and linear duplexes. In transfection only about 15% of the phage DNA is efficiently taken up by the competent cells. After entry of phage DNA into wild-type cells in transfection the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. These presumably concatamer forms are generated by recombination. In strain rec-1 the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in the wild-type cells. Since rec-1 is transfected with much lower efficiency than the wild-type our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for efficient transfection. These results also show that although phage N3 codes for its own recombination system it cannot operate in the early stages of transfection and succesful transfection is entirely dependent upon the host recombination system.     

Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.     

Production of infectious duck hepatitis B virus in a human hepatoma cell line.

The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks.     

Frozen and thawed bacteria as recipients of isolated phages and plasmid DNA]

Bacteria subjected to freezing and thawing are effective recipients of phage 1 phi 7 DNA, lambda DNA, and plasmid pMB9 DNA. The effectiveness of transfection and plasmid transformation of frozen and thawed bacteria is determined by the joint action of 3 factors: 1) the conditions of freezing and thawing of a recipient and DNA mixture with freezing carried out at a rate of 400 degrees C/min to--76 degrees C or--196 degrees C and with subsequent thawing at 42 degrees C; 2) a transitory character of recipient competence preservation in respect of phage and plasmid DNA; 3) the degree of recipient cryolability depending, in particular, on the genotype of the recipient. The maximum indices of transfection effectiveness and plasmid transformation have been obtained with bacterial concentration equal to 1--5 X 10(9) cells/ml, phage and plasmid DNA concentration equal to 0.05--0.5 mcg/ml in the reaction mixture containing 0.5--1% of Spofa bactopeptone, PH 7.4--7.6.     

Biological characteristics of a type I restriction-modification system in Staphylococcus aureus.

Two restriction-modification systems, S1 and S2, are present in Staphylococcus aureus RN450 (S. Iordanescu and M. Surdeanu, J. Gen. Microbiol., 96:277-281, 1976). System S2 affects phage multiplication after both infection and transfection. Unmodified plasmid and chromosomal DNAs are also not expressed following transduction and transformation into a restrictive host. Restricted phages are, however, capable of conferring phage-mediated competence, although the state of competence does not affect the restriction-modification system. The restricting activity of system S2 is inactivated by heat treatment of the cells. An enzymatic activity that restricts unmodified phage DNA in the presence of ATP, Mg2+, and S-adenosylmethionine was recovered from cell-free extracts of a strain RN450 derivative.     

Transfection of Staphylococcus aureus with Bacteriophage Deoxyribonucleic Acid

Staphylococcus aureus cells of strain 8325 (N) are competent for phage deoxyribonucleic acid (DNA) when harvested in the early exponential growth phase. Phenotypic expression of the competence requires divalent cations, and calcium ions are most effective. Treatment of phage DNA with deoxyribonuclease completely destroys infectivity and heat-denaturated DNA is not infectious. The highest frequency of transfection is around 10(4) plaque-forming units per mug of DNA.     

Initiation and maintenance of persistent infection by respiratory syncytial virus.

Propagation of cells infected with temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus at nonpermissive temperature (39 degrees C) resulted in cytolytic, abortive, or persistent infection, depending on the mutant used to initiate infection. Five mutants from complementation group B produced cytolytic or abortive infections, whereas a single mutant (ts1) from group D and a noncomplbmenting mutant produced persistent infections. The persistently infected culture initiated by mutant ts1 (RS ts1/BS-C-1) has been maintained in serial culture for greater than 100 transfers, and infectious-center assays and immunofluorescent staining indicated that all cells harbored the RS virus genome. RS ts1/BS-C-1 cultures were resistant to superinfection by homologous and some heterologous viruses, and interferon-like activity against some heterologous viruses was present in the culture medium. Small amounts (0.002 to 0.2 PFU/cell) of infectious virus were present in the culture fluid, but autointerfering defective particles were not detected. This released virus formed small plaques and produced persistent infection of BS-C-1 cells at 37 degrees C. The RS ts1/BS-C-1 cells contained abundant RS virus antigen internally, but little at the surface, although the cells showed enhanced agglutinability by concanavalin A. Nucleocapsids and the 41,000-molecular-weight nucleoprotein were present in extracts of both nucleated and enucleated cells. No infectious RS virus was obtained by transfection of DNA from RS tsl/BS-C-1 cells to susceptible BS-C-1 or feline embryo cells under conditions allowing efficient transfection of a foamy virus proviral DNA. It was concluded that persistent infection was maintained in part by a non-ts variant of RS virus partially defective in maturation. The karyotype of the RS ts1/BS-C-1 culture differed from that of unifected cells.     

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.     

Release of a special fraction of the outer membrane from both growing and phage T4-infected Escherichia coli B.

Growing Escherichia coli release envelope material into the medium. Upon infection with T4 phage increased amounts of this material are released and at a greater rate. In order to determine whether both inner and outer membranes are present in this material, and whether the material released by growing cells differs from that released by infected cells, we have examined the protein composition of envelope released by growing and T4-infected E. coli B. Our results show: (a) the protein composition of envelope released from growing or infected cells is similar, (b) the proteins present are representative of the outer membrane, (c) the major outer membrane protein of E. coli B, protein II, is deficient in the released material. We therefore conclude that the envelope material released from growing or infected E. coli represents a special fraction of the outer membrane. This finding is discussed in relation to outer membrane structure and function. In addition, data are presented on the differing outer membrane protein composition of substrains of E. coli B obtained from different laboratories.     

Recovery of competence in calcium-limited Azotobacter vinelandii.

Azotobacter vinelandii cells required 0.5 mM calcium in the iron-limited competence induction medium. This requirement also was fulfilled by strontium, but not by magnesium. Cells pregrown in competence medium lacking calcium rapidly recovered competence with the addition of 0.5 mM calcium, provided they were suspended in the growth supernatant. A 60,000-dalton glycoprotein (pI 5.10) present in competent or incompetent culture supernatants participated in calcium-mediated competence recovery. Cells grown in calcium-limited medium appeared to have leaky cell envelopes and released a diverse array of proteins into the culture supernatant and into distilled water washes of the cells, seven of which appeared to be more dominant in competent cells. Two distilled water washes of cells grown in calcium-limited medium did not prevent calcium-mediated recovery of competence in the culture supernatant. Four to six distilled water washes removed a competence-specific protein (pI 5.19) and prevented calcium-mediated recovery of competence in the culture supernatant.     

Thymidine nucleotide synthesis and catabolism by CHO cells and their changes during the cell cycle

At 0°C, CHO cells efficiently incorporated [³H]thymidine into the nucleotide fraction, but not into DNA. Upon reincubation of asynchronous cultures at 37°C, 15–25% of the radioactivity contained in the cellular nucleotide fraction was released, in the form of thymidine, into the culture medium. At 0°C, however, radioactivity of the nucleotide fraction was retained within the cells. Similarly, dTMP phosphatase (EC 3.1.3.35) in cell extracts was active at 37°C, but not at 0°C, whereas thymidine kinase (EC 2.7.1.21) was active at both temperatures. If synchronous cultures in Gl phase were prelabeled at 0°C and reincubated at 37°C, almost all radioactivity in the nucleotide fraction was released into the medium, whereas in S-phase cultures nearly all radioactivity of the nucleotide fraction was incorporated into DNA. In synchronous S-phase cultures treated with hydroxyurea, radioactivity in the nucleotide fraction was released into the medium at a rate considerably lower than that observed for Gl-phase cells. Rates of endogenous synthesis of thymidine nucleotides were calculated from changes of cellular thymidine nucleotide content, incorporation of thymidine nucleotides into DNA and release of thymidine into the medium during reincubation of prelabeled cultures in thymidine-free medium. The results obtained (see Table III) reveal marked differences between Gl and S phases with respect to the determinants of thymidine nucleotide metabolism.     

Bacteriophage T4D receptors and the Escherichia coli cell wall structure: role of spherical particles and protein b of the cell wall in bacteriophage infection.

The nature of the interaction of bacteriophage T4D and the outer cell wall of its host, Escherichia coli B, has been investigated. Bacteria with altered or modified cell walls have been obtained by two different growth procedures: (i) growth in high osmolarity medium or (ii) growth in broth in the presence of divalent heavy metal ions. When these altered host cells were washed and subsequently added to regular growth medium, they interacted with added phage particles, but successful infection did not occur. Most of the phage particles released from these treated cells were observed to have full heads and an altered tail structure. The altered phage tails had contracted sheaths and unusual pieces of the bacterial cell wall attached to the distal portion of the exposed phage tail tube. Phage released from bacteria grown in the high osmolarity medium had attached cell wall pieces of two major types, these pieces being either 40 or 21 nm in diameter. The smaller-type cell wall pieces (21 nm) were formed by three spheres each measuring 7 nm in diameter. Phage particles released from cells previously exposed to the divalent metal ions had only one 7-nm cell wall sphere attached to the distal end of the tail tube. It was found that these 7-nm spheres (i) are normal components of the cell wall and are morphologically similar to endotoxin, (ii) are held in place on the cell wall by a component of the cell wall called protein b, and (iii) are most likely the site of penetration of the phage tail tube through which the phage DNA enters the host cell.